In genetic research or in the diagnosis of infectious diseases an important task is the determination and classification of the nucleotide sequences at a particular region of the genome where it falls into two categories, which govern important features of the phenotype. The prime example of such a situation is the cleavage site of the avian influenza virus (AIV) haemagglutinin precursor protein (HA0), which is of a kind characteristic of either a low- or a high-pathogenic virus. The HA0 cleavage sites of the highly pathogenic AIV contain multiple basic amino acid side chains with the minimal motif R/K-X-R/K-R↓G and have so far only been found for H5 and H7 subtypes1. In fact, a large number of viruses have been shown to possess surface proteins for which post-translational cleavage is required for activation of infectivity2, including important pathogens such as human immunodeficiency virus type 1, the filoviruses Ebola and Marburg and flaviviruses, such as the yellow fever virus. Many of these viruses possess cleavage site motifs similar to those of the avian influenza viruses and there are several viruses for which correlations between pathogenicity and cleavage properties have been demonstrated1,2.
On the basis of the codon representation, the AIV high-pathogenic cleavage site motif can be represented by more than 18,000 distinct sequences at the RNA level. Several hundred of these possible sequences have been discovered in different H5 and H7 subtype viruses and novel viruses with previously unknown cleavage site sequences are constantly emerging. For this reason, attempts to probe the cleavage site in PCR applications have been limited to subsets of influenza viruses3-6 and current standard procedures for molecular testing of AIV pathogenicity involve nucleotide sequencing of the cleavage site3,7. Thus, single tube experiments for AIV pathotyping, able to avoid cumbersome, time consuming and technically demanding nucleotide sequencing, for rapid screening and diagnostic applications, must be able to interrogate the sample in a highly multiplexed way.
A major problem of differentiating large sets of genomic sequences is to devise multiplex amplification methods8 for obtaining homogeneous amplification of all recognized target segments and to suppress interactions between the primers.
Attempts have been made to suppress interactions betweens primers, e.g. by using the HANDS (Homo-tag Assisted Non-Dimer System) technology, in which chimerical primers (sequence recognizing primers tagged with a universal sequence) are present in relatively low concentration while universal primers targeting the complement of universal tags present on the chimerical primers, which in these scheme effectively brings the amplification to detectable levels, are at higher concentrations9. Since the same universal tag sequences extends both the forward and reverse primer of each chimerical primer pair, the ends of the amplification products will be self-complementary after a few PCR cycles. For the short, target independent, primer dimerization products the formation of intramolecular “panhandle” structures will be favoured, which will protect the 3′-end and prevent further amplification of the primer dimers9. The suppression of primer dimers will allow higher multiplexing.
In a related scheme10, which adapts the PCR suppression concept11 for multiplex detection, a universal primer target (adaptor) is ligated to the ends of the genomic DNA. The multiplex amplification is achieved by using one primer, common for all multiplex reactions, targeting the adaptor region and one target specific primer for each multiplex component reaction. Two mechanisms are proposed to explain the multiplex efficiency archived with this method10. First, the reduction of the number of primers used in the reaction, since the adaptor primer is identical for all reaction, reduces unwanted primer interactions. Secondly, spurious amplification brought about by the adaptor primer alone will produce amplicons with self-complementary ends and hence will form panhandle structures whose further amplification will be suppressed in the same way as proposed for the primer dimers in the HANDS method9.
Subsequently, the use of universal primers have found many uses in multiplex methods such as multiplex microarray enhanced PCR12, Templex PCR13, nested patch PCR14 but also in ligation based multiplex amplification methods such as multiplex ligation-dependent probe amplification15 and assays based on sequence tagged molecular inversion probes16.
Despite the efforts made in the art there is still a need for novel methods that provide homogeneous amplification of all recognized target segments in large sets of genomic sequences.